去B链羧端九肽胰岛素的制备和性质

去B链C端八肽胰岛素(DOI)经羧肽酶B作用除去B_(22)-精氨酸,产物经SP-SephadexC25分离以后得到电泳均一的去B链C端九肽胰岛素(DNI)。从CD光谱可以看出,在水溶液中DNI的构象比DOI更松散。DNI与人胎盘细胞膜胰岛素受体的结合能力很低,但却比DOI高一些。     

一种筛选单体速效胰岛素的简便方法

通过基因突变方法制备的单体速效胰岛素Lispro Insulin已上市用于治疗糖尿病,如何利用简便快速的方法研究获得新的单体速效胰岛素成为研究的热点。以Lispro Insulin为模型,利用猪胰岛素的胰蛋白酶酶切大片段(DOI,去B链C端八肽胰岛素)和化学合成的八肽,通过胰蛋白酶的酶促合成方法为筛选新的单体速效胰岛素提供了新的途径。结果显示,酶促合成得到的95％纯度的Lispro Insulin具备了单体速效胰岛素的不自身聚合的特点。     

B 链羧端去八肽胰岛素的核磁共振研究

用~1H 核磁共振(简称NMR)卷积差谱和温度差谱技术对胰岛素与去八肽胰岛素(简称DOI)局部溶液构象进行了比较研究。通过二者NMR 谱的比较发现胰岛素和DOI 谱的CH_3峰区存在明显差异。DOI 三维结构的屏蔽效应明显降低;以缬氨酸、亮氨酸、异亮氨酸CH_3组成的峰区变尖、变窄、接近自由蜷曲状态蛋白质谱。高场区0.42ppm 的峰肩为A_2-异亮氨酸在三维结构中受邻近A_(19)-酪氨酸屏蔽影响的高场位移峰,该峰在DOI 谱中消失。因此暗示着A 链头尾密切联系着的三维结构遭到破坏。用温度差谱简化NMR 谱。比较了胰岛素与DOI 二者随温度上升构象变化的过程以及各自对热的不稳定性,表明DOI 分子在溶液中还保存一定的特定构象;但结构相当松散,热不稳定性增加。胰岛素经去B 链羧端八肽后局部构象将有明显变化,可能导致胰岛素某种特定空间结构受到破坏。这也可能是DOI 分子生物活性丧失的一个重要原因。     

B链羧端去八肽胰岛素的核磁共振研究

用~1H核磁共振(简称NMR)卷积差谱和温度差谱技术对胰岛素与去八肽胰岛素(简称DOI)局部溶液构象进行了比较研究。通过二者NMR谱的比较发现胰岛素和DOI谱的CH_3峰区存在明显差异。DOI三维结构的屏蔽效应明显降低;以缬氨酸、亮氨酸、异亮氨酸CH_3组成的峰区变尖、变窄、接近自由蜷曲状态蛋白质谱。高场区0.42ppm的峰肩为A_2-异亮氨酸在三维结构中受邻近A_(19)-酪氨酸屏蔽影响的高场位移峰,该峰在DOI谱中消失。因此暗示着A链头尾密切联系着的三维结构遭到破坏。用温度差谱简化NMR谱。比较了胰岛素与DOI二者随温度上升构象变化的过程以及各自对热的不稳定性,表明DOI分子在溶液中还保存一定的特定构象;但结构相当松散,热不稳定性增加。胰岛素经去B链羧端八肽后局部构象将有明显变化,可能导致胰岛素某种特定空间结构受到破坏。这也可能是DOI分子生物活性丧失的一个重要原因。     

胰岛素B链N端和C端缩短对其与胰岛素抗体结合力的影响

本文研究了B链N端和C端缩短的若干胰岛素类似物与胰岛素抗体的结合能力。结果表明:去B链C端五肽胰岛素(DPI)分子中B_1-Phe去除后其与胰岛素抗体结合力明显下降,这与去B_1-Phe胰岛素与胰岛素抗体结合力下降的趋势相似;去B链C端六肽胰岛素(DHI)与胰岛素抗体结合力与DPI非常接近,都为胰岛素的70%左右。而去B链C端七肽胰岛素(DHPI)与胰岛素抗体结合力与去B链C端六肽胰岛素(DHI)相比,其结合力下降了一个数量级。说明胰岛素B_1-和B_(24)-Phe残基对组成和维持胰岛素分子的抗原决定簇起着重要作用。去B链九肽胰岛素(DNI)与胰岛素抗体的结合力与去B链C端八肽胰岛索(DOI)及DHPI相似。本文对上述结果进行了讨论。     

去B链羧端七肽人胰岛素的分离纯化及性质研究

在大肠杆菌温度诱导体系中以非融合方式进行去Ｂ链羧端七肽人胰岛素原基因的表达,获得去Ｂ链羧端七肽人胰岛素原,表达产物占细胞总蛋白量的１３％,表达产物经ＳｅｐｈａｄｅｘＧ－５０柱层析分离及胰蛋白酶和羧肽酶Ｂ的酶促转化等步骤,可得到纯度达９４％以上的去Ｂ链羧端七肽人胰岛素,其氨基酸组成与预期值相符,受体活性是标准猪胰岛素的１％．     

胰岛素片段与胰岛素抗体的相互作用

本文研究了若干胰岛素片段及去B链C端八肽胰岛素(DOI)、A链、B链与胰岛素抗体的结合能力。结果表明N 端区域片段保留部分胰岛素的免疫活力,而C 端片段除“工”字肽的免疫活力比A 链略高外,其他片段(B_(26～30),B_(22～26))均和A 链类似,没有胰岛素的免疫活力。以上结果进一步说明在免疫活力方面,胰岛素分子的N 端区域比C 端重要。同样,B 链可能比A 链重要。     

胰岛素片段与胰岛素抗体的相互作用

本文研究了若干胰岛素片段及去B链C端八肽胰岛素(DOI)、A链、B链与胰岛素抗体的结合能力。结果表明N端区域片段保留部分胰岛素的免疫活力,而C端片段除“工”字肽的免疫活力比A链略高外,其他片段(B_(26～30),B_(22～26))均和A链类似,没有胰岛素的免疫活力。以上结果进一步说明在免疫活力方面,胰岛素分子的N端区域比C端重要。同样,B链可能比A链重要。     

去B链羧端三肽人胰岛素的分离纯化及其性质研究

将去Ｂ链羧端三肽人胰岛素原基因克隆到表达质粒ｐＢＶ２２０上,在大肠杆菌系统中经温度诱导表达,表达产物占细胞总蛋白量的１２％,表达产物经ＳｅｐｈａｄｅｘＧ－５０柱层析分离以及胰蛋白酶和羧肽酶Ｂ的酶促转化等步骤,可得到去Ｂ链羧端三肽人胰岛素,其纯度达９３％,其氨基酸组成与预期值相符,但其受体结合活性仅是标准猪胰岛素的４５％．     

胰岛素类似物的直线聚合

与胰岛素相似,去B链羧端五肽胰岛素(DPI)及去B链羧端八肽胰岛素(DOI)的酸性溶液于沸水浴中加热,会转变成粘度很高的溶液,该溶液具有流动双折射性质。于电子显微镜下观察,看到该两种类似物的纤维。这些纤维与胰岛素纤维相似,经常由几根微丝绞合成螺旋状。微丝的直径大约是30,与胰岛素微丝的直径相当。上述现象说明,这些类似物的直线聚合过程与胰岛素者是类同的。因此胰岛素B链羧端肽段并非胰岛素直线聚合所必须。     

Composition of Mammalian Eumelanins: Analyses of DHICA-Derived Units in Pigments From Hair and Melanoma Cells

The proportions in which two eumelanin monomers, namely 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), compose the eumelanin polymer are believed to determine properties of the pigment including its color. These proportions are, however, not well elucidated for naturally occurring eumelanins, largely because of methodological difficulties. In this study we estimate the content of DHICA-derived units in mammalian eumelanins using a combination of two analytical techniques: 1) quantitation of DHICA-derived eumelanin by measuring the yield of pyrrole-2,3,5-tricarboxylic acid (PTCA index) and 2) spectrophotometrical quantitation of total (DHI + DHICA) eumelanin at 350 nm (A₃₅₀ index). The ratio of PTCA/A₃₅₀ measured for melanins synthesized from DHI and DHICA mixed in various molar proportions correlates well with the content of DHICA in synthetic polymers. Using this relationship as a standard curve we estimated the proportion of DHICA-derived units in mammalian eumelanins from hair and melanoma cells and found it to be much higher in rodent pigments (58.8%-98.3%; two species, mouse and hamster were examined) as compared to human eumelanins (19.2%-41.8%; one Caucasian and one Oriental individual were examined). No relationship between proportion of DHICA-derived units in eumelanin and hair color is found. The latter seems to be determined predominantly by the ratio of pheo- to eumelanin synthesis.     

Active involvement of PKC for insulin-mediated rates of muscle protein synthesis in Zucker rats

A recent report from our group demonstrated that insulin facilitates muscle protein synthesis in obese Zucker rats. The purpose of this study was to determine whether PKC, a probable modulator of insulin signal transduction and/or mRNA translation, has a role in this insulin-mediated anabolic response. In the first portion of the study, gastrocnemius muscles of lean and obese Zucker rats (n = 5-7 for each phenotype) were bilaterally perfused with or without insulin to assess cytosolic and membrane PKC activity. Limbs perfused with insulin demonstrated greater PKC activity in both lean and obese Zucker rats (P < 0.05) compared with no insulin, but overall activity was greater in obese animals (by approximately 27% compared with lean, P < 0.05). To determine whether PKC plays a role in muscle protein synthesis, hindlimbs (n = 6-8 for each phenotype) were bilaterally perfused with or without insulin and/or GF-109203X (GF; a PKC inhibitor). The presence of GF did not influence the rates of insulin-mediated protein synthesis in gastrocnemius muscle of lean Zucker rats. However, when obese rats were perfused with GF (P < 0.05), the effect of insulin on elevating rates of protein synthesis was not observed. We also used phorbol 12-myristate 13-acetate (TPA, a PKC activator; n = 5-7 for each phenotype) with and without insulin to determine the effect of PKC activation on muscle protein synthesis. TPA alone did not elevate muscle protein synthesis in lean or obese rats. However, TPA plus insulin resulted in elevated rates of protein synthesis in both phenotypes that were similar to rates of insulin alone of obese rats. These results suggest that PKC is a modulator and is necessary, but not sufficient, for insulin-mediated protein anabolic responses in skeletal muscle.     

Inactivation of phosphorylase is a major component of the mechanism by which insulin stimulates hepatic glycogen synthesis.

Multiple signalling pathways are involved in the mechanism by which insulin stimulates hepatic glycogen synthesis. In this study we used selective inhibitors of glycogen synthase kinase-3 (GSK-3) and an allosteric inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a, to determine the relative contributions of inactivation of GSK-3 and dephosphorylation of phosphorylase a as alternative pathways in the stimulation of glycogen synthesis by insulin in hepatocytes. GSK-3 inhibitors (SB-216763 and Li+) caused a greater activation of glycogen synthase than insulin (90% vs. 40%) but a smaller stimulation of glycogen synthesis (30% vs. 150%). The contribution of GSK-3 inactivation to insulin stimulation of glycogen synthesis was estimated to be less than 20%. Dephosphorylation of phosphorylase a with CP-91149 caused activation of glycogen synthase and translocation of the protein from a soluble to a particulate fraction and mimicked the stimulation of glycogen synthesis by insulin. The stimulation of glycogen synthesis by phosphorylase inactivation cannot be explained by either inhibition of glycogen degradation or activation of glycogen synthase alone and suggests an additional role for translocation of synthase. Titrations with the phosphorylase inactivator showed that stimulation of glycogen synthesis by insulin can be largely accounted for by inactivation of phosphorylase over a wide range of activities of phosphorylase a. We conclude that a signalling pathway involving dephosphorylation of phosphorylase a leading to both activation and translocation of glycogen synthase is a critical component of the mechanism by which insulin stimulates hepatic glycogen synthesis. Selective inactivation of phosphorylase can mimic insulin stimulation of hepatic glycogen synthesis.     

Preparation of eumelanin-related metabolites 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylic acid, and their O-methyl derivatives

5,6-Dihydroxyindole (5,6DHI) and 5,6-dihydroxyindole-2-carboxylic acid (5,6DHI2C) are ultimate precursors of the black melanin, eumelanin. These indolic metabolites and their O-methyl derivatives are excreted in urine of melanoma patients at high levels and of healthy persons at low levels. We describe here a simplified procedure for preparing milligram to subgram quantities of 5,6DHI and 5,6DHI2C and their O-methyl derivatives. Dopachrome generated in situ by ferricyanide oxidation of dopa at pH 6.5 underwent spontaneous decarboxylation to give 5,6DHI in 40% isolation yield, while treatment of dopachrome with alkali at pH 13 afforded 5,6DHI2C in 38% isolation yield. Two isomeric O-methyl derivatives of 5,6DHI were prepared by treatment with diazomethane, while those of 5,6DHI2C were prepared by treatment with diazomethane followed by alkaline hydrolysis of the methyl esters. 5,6DHI and 6-hydroxy-5-methoxyindole were also obtained by heating the corresponding carboxylic acids in decalin. 5-Hydroxy-6-methoxyindole and 6-hydroxy-5-methoxyindole-2-carboxylic acid could also be prepared by debenzylation of the commercially available O-benzyl derivatives.     

Dopaquinone redox exchange with dihydroxyindole and dihydroxyindole carboxylic acid

A pulse radiolytic investigation has been conducted to establish whether a redox reaction takes place between dopaquinone and 5,6-dihydroxyindole (DHI) and its 2-carboxylic acid (DHICA) and to measure the rate constants of the interactions. To obviate possible confounding reactions, such as nucleophilic addition, the method employed to generate dopaquinone used the dibromide radical anion acting on dopa to form the semiquinone which rapidly disproportionates to dopaquinone. In the presence of DHI the corresponding indole-5,6-quinone (and/or tautomers) was also formed directly but, by judicious selection of suitable relative concentrations of initial reactants, we were able to detect the formation of additional indolequinone from the redox exchange reaction of DHI with dopaquinone which exhibited a linear dependency on the concentration of DHI. Computer simulation of the experimental time profiles of the absorption changes showed that, under the conditions chosen, redox exchange does proceed but not quite to completion, a forward rate constant of 1.4 x 10(6)/M/s being obtained. This is in the same range as the rate constants previously established for reactions of dopaquinone with cyclodopa and cysteinyldopa. In similar experiments carried out with DHICA, the reaction more obviously does not go to completion and is much slower, k (forward) =1.6 x 10(5)/M/s. We conclude that, in the eumelanogenic pathway, DHI oxidation may take place by redox exchange with dopaquinone, although such a reaction is likely to be less efficient for DHICA.     

Effect of main ingredients of Danhong Injection against oxidative stress induced autophagy injury via miR-19a/SIRT1 pathway in endothelial cells

BackgroundAutophagy plays an important role in cellular homeostasis. Oxidative stress stimulated endothelial excessive autophagy has been proposed as a major risk factor for cardiovascular diseases (CVD). Danhong injection (DHI), the most prescribed traditional Chinese medicine for the treatment of CVD, has been shown to elicit vascular protective effects. However, its underlying mechanisms remain poorly defined. This study aimed to uncover the protective effects of DHI and its main bioactive components on autophagy injury of human umbilical vein endothelial cells (HUVECs) induced by H₂O₂ and reveal the possible mechanisms.MethodsHUVECs were treated with different concentrations of DHI or its components, after exposed to H₂O₂. The protective effects of DHI and its components in H₂O₂-induced HUVECs were examined via a cytotoxicity assay and western blot. Apoptosis was evaluated with flow cytometry. Autophagy flux was assessed by transmission electron microscopy and LC3 plasmid transfection. Besides, the role miR-19a and SIRT1 in DHI and components-mediated anti-autophagy responses were validated with inhibitors transfection.ResultsOur results showed that DHI and its components do have different effects on different aspects. In terms of HUVECs survival rate, Salvianolic acid B (Sal B) and danshensu (DSS) performed better than DHI, Hydroxysafflor yellow A (HSYA) and Tanshinone IIA (DST-IIA). As for the proliferation effect on HUVECs, only Sal B has the most obvious performance as same as 3MA. Besides, DHI and its components are sensitive and superior in regulating and balancing ROS concentration. Among the GSH/GSSG indicators, DSS and HSYA performed better. In terms of SOD content and apoptotic rate, the SOD level showed the opposite trend compared with H₂O₂ group. For the expression of LC3, Beclin-1 and P62, DHI and its components all had significant effects. When miR-19a or SIRT1 was inhibited, Sal B (0.5 μg/ml) can not decrease autophagy-related protein effectively.ConclusionDHI and its components all had anti-autophagy effects. And Sal B (0.5 μg/ml) inhibited HUVECs autophagy via miR-19a/SIRT1 pathway.     

Eumelanin buildup on the nanoscale: aggregate growth/assembly and visible absorption development in biomimetic 5,6-dihydroxyindole polymerization

Establishing structure-property relationships in the black insoluble eumelanins, the key determinants of human pigmentation and skin photoprotective system, is a considerable conceptual and experimental challenge in the current drive for elucidation of the biological roles of these biopolymers and their application as advanced materials for organoelectronics. Herein, we report a new breakthrough toward this goal by the first detailed investigation on the nanoscale level of the oxidative polymerization of 5,6-dihydroxyindole (DHI), a model process of eumelanin synthesis. On the basis of a combined use of spectrophotometry, dynamic light scattering (DLS), and small-angle neutron scattering (SANS) investigations, it was possible to unveil the dynamics of the aggregation process before precipitation, the key relationships with visible light absorption and the shape of fundamental aggregates. The results indicated a polymerization mechanism of the type: Polymer(n) + DHI(x) = Polymer(n+x), where DHI(x) indicates monomer, dimer, or low oligomers (x ≤ 5). During polymerization, visible absorption increases rapidly, reaching a plateau. Particle growth proceeds slowly, with formation of 2-D structures ～55 nm thick, until precipitation occurs, that is, when large aggregates with a maximum hydrodynamic radius (R(h)) of ～1200 nm are formed. Notably, markedly smaller R(h) values, up to ～110 nm, were determined in the presence of poly(vinyl alcohol) (PVA) that was shown to be an efficient aggregation-preventing agent for polymerizing DHI ensuring water solubilization. Finally, it is shown that DHI monomer can be efficiently and partially irreversibly depleted from aqueous solutions by the addition of eumelanin suspensions. This behavior is suggested to reflect oxidant-independent competing pathways of polymer synthesis and buildup via monomer conversion on the active aggregate surface contributing to particle growth. Besides filling crucial gaps in DHI polymerization, these results support the attractive hypothesis that eumelanins may behave as a peculiar example of living biopolymers. The potential of PVA as a powerful tool for solution chemistry-based investigations of eumelanin supramolecular organization and for technological manipulation purposes is underscored.     

Kinetics of G1 transit following brief starvation for serum factors

Growing fibroblasts such as 3T3 cells are well-known to enter a quiescent state (G0) after many hours of serum deprivation. They emerge from G0 upon readdition of serum and initiate DNA synthesis about 12 h later. In this paper, we analyzed the effects of brief periods of serum deprivation on the ability of cells in G1 to initiate DNA synthesis. Exponentially growing 3T3 fibroblasts were briefly deprived of serum and their progress into S phase was monitored by autoradiography of labeled nuclei. When 10% serum was added back to cultures deprived of serum for a few hours, the progress of G1 cells into S phase was delayed for intervals far in excess of the length of the serum deprivation. Longer serum starvations resulted in longer excess delays. Several transformed 3T3 derivatives were markedly less sensitive to this serum-induced G1 regression following deprivation. When 1 microgram/ml insulin (rather than 10% serum) was added back to the starved cultures, the G1 cells entered S phase immediately. Delay in S phase entry following serum readdition was completely prevented if insulin (and, to a lesser extent, EGF) was present during the starvation, was diminished if a lower serum concentration was used for readdition, and was partially abolished if 10% serum plus insulin was restored to the cultures. The above results, then, suggest that serum deprivation sensitizes the cells to an unidentified serum component which sets the cells back in G1, unless insulin is present to maintain the flow of cells into S.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.